Missense Mutations of the Pro65 Residue of PCGF2 Cause a Recognizable Syndrome Associated with Craniofacial, Neurological, Cardiovascular, and Skeletal Features  Peter D. Turnpenny, Michael J. Wright, Melissa Sloman, Richard Caswell, Anthony J. van Essen, Erica Gerkes, Rolph Pfundt, Susan M. White, Nava Shaul-Lotan, Lori Carpenter, G. Bradley Schaefer, Alan Fryer, A. Micheil Innes, Kirsten P. Forbes, Wendy K. Chung, Heather McLaughlin, Lindsay B. Henderson, Amy E. Roberts, Karen E. Heath, Beatriz Paumard-Hernández, Blanca Gener, Katherine A. Fawcett, Romana Gjergja-Juraški, Daniela T. Pilz, Andrew E. Fry  The American Journal of Human Genetics  Volume 103, Issue 5, Pages 786-793 (November 2018) DOI: 10.1016/j.ajhg.2018.09.012 Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 1 Facial Features of Individuals with PCGF2 Mutations Each individual is noted with the corresponding number used throughout the manuscript. Included on the top left of each cluster is the sex. Ages are: individual 1, 12 years; individual 2, 8.5 years; individual 3, 8 years; individual 4, 8 years; individual 5, 7 years (left), 4 years (right); individual 6, 9 years; individual 7, 7 years; individual 8, 6 years; individual 9, 2 years (left), 3 years (right); individual 10, 9 years; individual 11, 8 years; individual 12, 3 months. Consistent facial features include a broad forehead, long face, malar hypoplasia, small mouth, small palpebral fissures, periorbital fullness, prominent nose (particularly in older indiduals), and dysplastic, low-set ears. The American Journal of Human Genetics 2018 103, 786-793DOI: (10.1016/j.ajhg.2018.09.012) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 2 MRI Brain Abnormalities in Individuals with PCGF2 Mutations (A–E) Axial (FLAIR, T2 and T1), sagittal (T1), and MRA images from individual 3 at age 21 months. (F–H) Axial (FLAIR and T1) and sagittal (T1) images from individual 4 at age 21 months. (I and J) Axial FLAIR and MRA images from individual 5 at age 3 years and 3 months. (K–M) Axial (FLAIR, T2 and T1) images from individual 6 at age 5 years. (N and O) Axial (T2 and T1) images from individual 7 at age 10 months. (P–R) Axial (FLAIR and T2) and MRA images from individual 8 at age 37 months. (S and T) Axial T2 images from individual 10 at age 13 months. The images show patchy white matter hyperintensity in the T2 and FLAIR images (black arrows). The patches are scattered throughout the white matter but are consistently seen in the peri-atrial region. Other findings included enlargement of the lateral ventricles; increased anterior extra-axial fluid spaces (e.g., B, G, N, and S); bilateral polymicrogyria (yellow arrows, subtle in M); prominence of the perivascular spaces, including in the corpus callosum (red arrow, Q); coarctation of the frontal horns (blue arrow, S). MRA showed tortuosity (white arrows) of the internal carotid (E), (J), and vertebral (R) arteries. The American Journal of Human Genetics 2018 103, 786-793DOI: (10.1016/j.ajhg.2018.09.012) Copyright © 2018 American Society of Human Genetics Terms and Conditions

Figure 3 Localization of the PCGF2 Mutations (A) Schematic domains of PCGF2. The domains and motifs of PCGF2 (UniprotKB: P35227) are illustrated. These include a RING-type Zinc finger (residues 18–57), nuclear localization signal (81–95) and Proline/Serine-rich domain (242–344). The location of the Pro65 residue is marked by the red asterisk. Residue number is indicated in the scale below the illustration. (B) The PCGF2 mutations are located at the highly conserved Pro65 residue. ClustalW homology alignments for Human PCGF2 (residues 51-80) and a range of orthologs and paralogs. Orthologs include Human (NP_009075.1), Rhesus monkey (XP_001083817.1), Cow (NP_001137578.1), Mouse (NP_001156779.1), Rat (NP_001099306.1), Chicken (XP_003642857.1), Frog (NP_001025573.1), Lamprey (ENSPMAP00000007297.1), and Fruit fly (NP_523725.2). Paralogs include BMI1/PCGF4 (NP_005171.4), PCGF1 (NP_116062.2), PCGF3 (NP_006306.2), PCGF5 (NP_001243478.1), and PCGF6 (NP_001011663.1). Identical residues are indicated by dots. The blue bar highlights the position of the Pro65 residue. (C) Structural model of human PCGF2 (residues 5-101, modeled on template 2h0d). Two views of the model are shown in ribbon format, colored from blue, N-terminal, to red, C-terminal, with side-chains shown for Pro65, Arg64, and Lys62. Grey spheres represent bound zinc ions; interaction with RING1B is primarily mediated via residues in helices 1 and 3 (blue and orange, respectively). (D) The interaction between PCGF2 and histone H3/4 (modeled on template 4r8p). The predicted molecular surface of PCGF2 (left) is colored by electrostatic charge (blue, basic; red, acidic); histone chains from 4r8p are shown as green (H3.2) and gray (H4) ribbons, respectively; other chains of the complex have been omitted for clarity; note the basic patch of PCGF2 in contact with H3.2. (E) As (D), but showing surface charge for H3.2, with PCGF2 shown as a ribbon colored from N-terminal, blue to C-terminal, red; note the acidic patch of H3.2 lying opposite the basic patch of PCGF2. (F) As (E), but showing detail around the PCGF2/H3.2 interface; the regions shown are outlined by gray broken lines in part E; sidechains of PCGF2 Pro65, Arg64, and Lys62 (partially obscured) are shown in stick format. The American Journal of Human Genetics 2018 103, 786-793DOI: (10.1016/j.ajhg.2018.09.012) Copyright © 2018 American Society of Human Genetics Terms and Conditions